ES2601830T3 - Apolipoprotein A-1 resistant to oxidants and mimetic peptides - Google Patents

Abstract

An ApoA1 polypeptide for use in a method of treating cardiovascular disorders, the ApoA1 polypeptide, capable of stimulating cholesterol efflux in lipid-loaded cells, has an amino acid sequence of SEQ ID NO: 1, in which each amino acid X in each amino acid sequence of SEQ ID NO: 1 is phenylalanine.

Description

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DXLKAFYDKFFEKFKEFF- (SEQ ID NO: 9), DXFKAFYDKFFE KFKEFF- (SEQ ID NO: 10), DXLKAFYDKVFEKFKEAF- (SEQ ID NO: 11), DXLKAFYDKVFEKFKKDF-12 NOK-ID: ), DXLKAFYDKVAEKFKEFF- (SEQ ID NO: 14), DXLKAFYDKVFEKFKEFF- (SEQ ID NO: 15), EXLKLFYEKVLEKFKEAF- (SEQ ID NO: 16), EXLKAFYDKVAEKFKKK ID NOKEK- ID: 18), EXLKAFYDKVFEKFKEAF- (SEQ ID NO: 19), EXLKAFYDKVFEKLKEFF- (SEQ ID NO: 20), EXLKAFYDKVAEKFKEFF- (SEQ ID NO: 21), EXLKAFYDKVFEKFKEFK- (SEQ ID NOKALK) : 23), DXFKAFYEKVAEKLKEFF- (SEQ ID NO: 24), DXFKAFYEKFFEKFKEFF- (SEQ ID NO: 25), EXLKALYEKVAEKLKEAL- (SEQ ID NO: 26), EXLKAFYEKVAEKLKEAF- (SEQ Q ID NO: 27), 20 EXFKAFYEKVAEKLKEFF- (SEQ ID NO: 28), EXLKAFYEKVFEKFKEFF- (SEQ ID NO: 29), EXLKAFYEKFFEKFKEFF- (SEQ ID NO: 30), EXFKAFYEKFFEKFKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 32), 25 EFLKA XYEKVAEKLKEAX- (SEQ ID NO: 33), DFXKAXYDKVAEKLKEXX- (SEQ ID NO: 34), EFXKAXYEKVAEKLKEXX- (SEQ ID NO: 35), DKLKAFYDKVFEXD: 36) DKXKAVYDKFAEAFKEFL- (SEQ ID NO: 37), 30 EKLKAFYEKVFEXAKEAF- (SEQ ID NO: 38), EKXKAVYEKFAEAFKEFL- (SEQ ID NO: 39), DXLKAFVDKFAEKFKEAY- (SEQ ID NOKEA- (SEQ ID NOKEA-) ,

D-X-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F- (SEQ ID NO: 42),

35 EXLKAFVYEKVFKLKEFF- (SEQ ID NO: 43), DXLRAFYDKVAEKLKEAF- (SEQ ID NO: 44), EXLRAFYEKVAEKLKEAF- (SEQ ID NO: 45), DXLKAFYDRVAEKLKEAF- (SEQ ID NOK) (SEQ ID NOVEA- 47) ,

40 DXLKAFYDKVAERLKEAF- (SEQ ID NO: 48), EXLKAFYEKVAERLKEAF- (SEQ ID NO: 49), DXLKAFYDKVAEKLREAF- (SEQ ID NO: 50), EXLKAFYEKVAEKLREAF- (SEQ ID NOK) (SEQ ID NOVEA- 51) ,

45 EXLKAFYERVAERLKEAF- (SEQ ID NO: 53), DXLRAFYDKVAEKLREAF- (SEQ ID NO: 54), EXLRAFYEKVAEKLREAF- (SEQ ID NO: 55), DXLRAFYDRVAEKLKEAF- (SEQ ID NOF: 56) ,

50 DXLKAFYDKVAERLREAF- (SEQ ID NO: 58), EXLKAFYEKVAERLREAF- (SEQ ID NO: 59), DXLRAFYDKVAERLKEAF- (SEQ ID NO: 60), EXLRAFYEKVAERLKEAF- (SEQ ID NOKLDKAKDKDK: ),

55 DXLKAFYDKVAEKLKEFFPDX-LKAFYDKVAEKLKEFF (SEQ ID NO: 63), DXFKAFYDKVAEKLKEAFPDX-FKAFYDKVAEKLKEAF (SEQ ID NO: 64), DKLKAFYDKVFEXAKEAFPDK-LKAFYDKVFEXLKEAF (SEQ ID NO: 65), DKXKAVYDKFAEAFKEFLPDK-XKAVYDKFAEAFKEFL (SEQ ID NO: 66), DXFKAFYDKVAEKFKEAFPDX-FKAFYDKVAEKFKEAF (SEQ ID NO: 67),

60 D-X-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-P-D-X-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F (SEQ ID NO: 68), and D-X-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F-P-D-X-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F (SEQ ID NO: 69);

in which X is phenylalanine.

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The sequences mentioned above (SEQ ID NO: 4-SEQ ID NO: 69) are described in US Patent No. 7,144,862 B2 of Fogelman et al. (Hereinafter, the patent '862). The '862 patent refers to peptides used to improve one or more symptoms of atherosclerosis. The peptides described in the '862 patent include a tryptophan moiety in each moiety designated herein with an X. The synthetic peptides of the' 862 patent were designed to mimic the class A amphipathic helical motif (Segrest et al. ., (1990) Proteins: Structure, Function and Genetics 8: 103-117)) and are capable of associating with phospholipids and have many biological properties similar to those of human ApoA1.

It is also indicated that this list of ApoA1 mimetic peptides is not fully inclusive. Truncation of the aforementioned sequences, multimeric combinations (for example, varying from dimers to trimers, tetramers, 5 meters, 8 meters, or 10 meters) of the aforementioned sequences, conservative substitutions of the aforementioned sequences, and / or The aforementioned sequences comprising amino acid analogs are also contemplated using the teachings provided therein.

It will be noted that biologically functional equivalents, or even improvements, of ApoA1 mimetic polypeptides, can be prepared, generally using ApoA1 as a starting point. Modifications and changes of such a protein can be made in the structure and also a molecule with similar or otherwise desirable characteristics can be obtained. For example, certain amino acids may be substituted for other amino acids in the protein structure without appreciable loss of cholesterol efflux acceptor activity.

It should also be contemplated that one skilled in the art can further modify the ApoA1 amino acid sequences of the present invention by substitution, deletion or addition of at least one amino acid, and that these additional modifications create biologically functional equivalents for resistant ApoA1 polypeptides. to oxidants These substitutions do not substantially inhibit the ability of the modified ApoA1 to stimulate cholesterol efflux from charged lipid cells. As used herein, "substantially inhibit" or "inhibition" includes any measurable reproducible reduction in the ability of a modified ApoA1 polypeptide to stimulate cholesterol efflux, from charged lipid cells.

The skilled person also understands that, inherent in the definition of a "biologically functional equivalent" protein or polypeptide, is the concept that there is a limit to the number of changes that can be made within a defined part of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent proteins and peptides are therefore defined herein as the proteins and peptides in which certain, not most or all, of the amino acids can be substituted. Of course, a plurality of different proteins / peptides with different substitutions can be easily prepared and used according to the invention.

Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, for example, and hydrophobia, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side chain substituents reveals that arginine, lysine and histidine are all positively charged moieties; that alanine, glycine and serine are all of a similar size. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine are defined herein as biologically functional equivalents.

Following the procedures indicated in the application published by Alton et al. (WO83 / 04053), genes encoding microbial expression of polypeptides having primary conformations that differ from those specified herein can be easily designed and manufactured. in terms of the identity or location of one or more remains (for example, substitutions, additions and terminal and intermediate deletions). Alternatively, cDNA and genomic gene modifications can be easily achieved by well-known site-directed mutagenesis techniques and used to generate ApoA1 analogs and derivatives. Such products could share at least one of the biological properties of the modified oxidant-resistant ApoA1 but could be differentiated into others.

ApoA1 mimetic polypeptides can be full length human ApoA1 peptide fragments. The ApoA1 described herein are biologically functional equivalents with respect to the ApoA1 polypeptides described above in at least one aspect that includes, but is not limited to, the presentation of cholesterol efflux and improvement of one or more symptoms of a inflammatory condition ApoA1 fragments may consist of about 5 to about 50 amino acids and include at least one tryptophan moiety, in which the tryptophan moiety is substituted with an oxidant resistant amino acid.

It will be noted that, as with the ApoA1 mimetic polypeptide, modifications and changes in the amino structure and sequence of the ApoA1 fragments can be made and also a molecule having similar or otherwise desirable characteristics can be obtained. For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of cholesterol efflux presentation.

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The ApoA1 mimetic polypeptides of the present invention may also include a protecting group coupled to the amino terminal and / or the carboxyl terminal of the polypeptides. The term "protecting group" refers to a chemical group that, when attached to a functional group in an amino acid (for example, a side chain, alpha amino group, an alpha carboxyl group, etc.) blocks or masks the properties of That functional group. Examples of amino terminal protecting groups include, but are not limited to acetyl, or amino groups. In such an embodiment, the first to four amino acid residues at the N-terminal and / or C-terminal end of the polypeptides described herein may be substituted with one or more amino acid residues, or one or more segments. of peptide, which are known to transmit stability to regions of the α-helical secondary structure (moieties or segments of "terminal protection" or "protecting groups"). Such terminal protection moieties and segments are well known in the art (see, for example, Richardson and Richardson, 1988, Science 240: 16481652; Harper et al., 1993, Biochemistry 32 (30): 7605-7609; Dasgupta and Bell , 1993, Int. J. Peptide Protein Res. 41: 499-511; Seale et al., 1994, Protein Science 3: 1741-1745; Doig et al., 1994, Biochemistry 33: 3396-3403; Zhou et al. , 1994, Proteins 18: 1-7; Doig and Baldwin, 1995, Protein Science 4: 1325-1336; Odaert et al., 1995, Biochemistry

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12820-12829; Petrukhov et al., 1996, Biochemistry 35: 387-397; Doig et al., 1997, Protein Science 6: 147-155). Alternatively, the first of one to four N-terminal and / or C-terminal amino acid residues of the polypeptides described herein can be substituted with peptidomimetic moieties that mimic the structure and / or properties of terminal protection moieties or segments. . Examples of terminal protection mimetics are well known in the art, and are described, for example, in Richardson and Richardson, 1988, Science 240: 16481652; Harper et al., 1993, Biochemistry 32 (30): 7605-7609; Dasgupta and Bell, 1993, Int. J. Peptide Protein Res. 41: 499-511; Seale et al., 1994, Protein Science 3: 1741-1745; Doig et al., 1994, Biochemistry 33: 3396-3403; Zhou et al., 1994, Proteins 18: 1-7; Doig and Baldwin, 1995, Protein Science 4: 1325-1336; Odaert et al., 1995, Biochemistry

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12820-12829; Petrukhov et al., 1996, Biochemistry 35: 387-397; Doig et al., 1997, Protein Science 6: 147-155).

The ApoA1 mimetic polypeptides of the present invention can be purified and isolated. The term "unified and isolated" herein means substantially free of unwanted substances so that the present polypeptides of modified ApoA1 mimetics are useful for stimulating cholesterol efflux from cells loaded with lipid. For example, a modified recombinant human ApoA1 mimetic polypeptide can be had substantially free of other human proteins or pathological agents. Polypeptides are also characterized by being a product of mammalian cells, or the product of chemical synthesis or prokaryotic or eukaryotic host expression procedures (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by genetic synthesis. Expression products in yeast host cells (e.g. Saccharomyces cerevisiae) or prokaryotes (e.g., E. coli) are free of association with any mammalian protein. Expression products in vertebrates (for example, non-human mammalian cells (eg, COS or CHO) and avian) are free from association with any human protein. Depending on the host employed, and other factors, the polypeptides of the invention may be glycosylated with mammalian carbohydrates or other eukaryotic carbohydrates or they may not be glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue (at position -1 with respect to the first amino acid residue of the polypeptide).

The peptides of the invention can be purified by techniques known in the art such as reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify a particular peptide will depend, in part, on the synthesis strategy and factors, such as net loading, hydrophobia, hydrophilicity, etc., and are evident to people with experience in the field. Multimeric branched peptides can be purified, for example, by ion exchange or size exclusion chromatography.

For purification by affinity chromatography, any antibody that specifically binds to the peptide can be used. For the production of antibodies, various host animals, which include, but are not limited to rabbits, mice, rats, etc., can be immunized by injection with a peptide. The peptide can be attached to a suitable vehicle, such as BSA, by means of a side chain functional group or connectors attached to a side chain functional group. Various adjuvants can be used to increase the immune response, depending on the host species, including, but not limited to, Freund (complete and incomplete), mineral gels such as aluminum hydroxide, active surface substances such as lisolecitin, pluronic polyols, polyanions. , peptides, oil emulsions, California limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Calmette-Guerin bacilli) and Corynebacterium parvum.

Monoclonal antibodies to a peptide can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but will not be limited to, the hybridoma technique originally described by Kohler and Milstein, 1975, Nature 256: 495-497, or Kaprowski, Pat. No. 4,376,110, the human B lymphocyte hybridoma technique) (Kosbor et al., 1983, Immunology Today 4: 72; Cote et al., 1983, Proc. Natl: Acad. Sci. USA 80 : 2026-2030); and EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). In addition, some techniques developed for the production of "chimeric antibodies" can be used (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al., 1984, Nature 312: 604 -608; Takeda et al., 1985, Nature 314: 452-454, Boss, US Pat. No. 4,816,397;

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for targeting host cell receptors in vivo), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553- 6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT Publication No. WO 88/09810, published December 15, 1988), cleavage agents activated by hybridization. (See, for example, Krol et al. (1988) BioTechniques 6: 958-976) or intercalating agents. (See, for example, Zon (1988) Pharm. Res. 5: 539-549). To this end, the oligonucleotides can be conjugated to another molecule, for example, a peptide, hybridization activated crosslinking agent, transport agent, hybridization activated cleavage agent, etc.

In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, a coding sequence can be linked to an adenovirus transcription / translation control complex, for example, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (for example, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the host peptide is infected. (For example, see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81: 3655-3659). Alternatively, the 7.5 K vaccinia promoter can be used, (see, for example, Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79: 74157419; Mackett et al., 1984 , J. Virol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79: 4927-4931).

Other expression systems for producing the polypeptides of the invention will be apparent to those with experience in the art. The DNA sequences described herein, which encode modified ApoA1 polypeptides are valuable for the information they provide regarding the mammalian protein amino acid sequence that I have so far not been available. In other words, DNA sequences can be useful for generating new and useful viral and circular plasmid DNA vectors, prokaryotic host cells follow new and useful transformed and transfected eukaryotes (including bacterial and yeast cells and mammalian cells developed in culture), and new and useful methods for the growth in culture of such host cells capable of expressing ApoA1 polypeptides resistant to modified oxidants and their related products.

Alternatively, no vector may be used to facilitate the relatively stable presence in the host. For example, homologous recombination can facilitate integration into a host genome. The nucleic acid may be placed within a pharmaceutically acceptable carrier to facilitate cellular absorption, such as a lipid solution vehicle (e.g., a charged lipid), a liposome, or polypeptide carrier (e.g., polylysine). A review article on gene therapy is Verma, Scientific American, November 1990, pages 68-84.

The desired nucleic acid can first be placed inside a cell, and the cell can be administered to a patient (such as a transplanted tissue) or the desired nucleic acid can be administered directly to the patient for absorption in vivo. The cells to be transferred to the receptor can be cultured using one or more factors that affect the growth or proliferation of said cells, such as SCF.

Nucleic acid molecules encoding a modified oxidant resistant ApoA1 mimetic conjugate, such as a fusion protein, can also be used in the invention. Such nucleic acids can be made by preparing a construct (for example, an expression vector) that expresses a ApoA1 mimetic fusion protein when introduced into a suitable host. For example, such a construction can be linked by ligation of a first polynucleotide encoding a modified oxidant-resistant ApoA1 mimetic protein capable of stimulating cholesterol efflux from cells loaded with lipid, fused in frame a second polynucleotide encoding another protein so that the expression of the construct in a suitable expression system produces a fusion protein.

ApoA1 mimetic fusion proteins can be easily prepared using molecular biology techniques. Any fusion protein may be designated and prepared using any of the therapeutic agents disclosed herein and those known in the art. Fusion protein technology is rapidly adapted to prepare fusion proteins in which the two parts are linked by a sequence of peptides that can be selectively cleaved. The use of recombinant DNA techniques to achieve such ends is currently a conventional practice for those skilled in the art. These procedures include, for example, in vitro recombinant DNA techniques, in vivo synthesis and recombination / genetic recombination techniques. In addition, DNA and RNA synthesis can be performed using an automated synthesizer.

The preparation of such a fusion protein usually involves the preparation of a first and second DNA coding region and the functional binding or binding of such regions, in frame, to prepare a single coding region encoding the fusion protein. desired. It is generally not believed to be particularly relevant which part of the construction is prepared as the N-terminal region or as the C-terminal region.

The present invention also relates to pharmaceutical compositions and / or formulations and to the use of such compositions in the treatment of hyperlipidemia, hypercholesterolemia, coronary heart disease, and atherosclerosis. The pharmaceutical compositions of the invention may include the ApoA1 mimetic polypeptide as the active ingredient and a pharmaceutically acceptable excipient suitable for administration and delivery.

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in vivo The pharmaceutical compositions will generally comprise an effective amount of modified ApoA1 polypeptides dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Combined therapeutic agents are also contemplated, and the same type of underlying pharmaceutical compositions can be used for both individual and combination medications.

The terms "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other unwanted creation reaction when administered to an animal, or a human being, if appropriate. Veterinary uses are included in the same manner within the invention and "pharmaceutically acceptable" formulations include formulations for both clinical and / or veterinary use.

As used herein, "pharmaceutically acceptable carrier" includes each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional means or agent is incompatible with the active substance, its use in therapeutic compositions is contemplated. Upon administration in humans, the preparations should meet the standards of sterility, pyrogeny, general safety and purity as required by the FDA Office of Biologics standards. Supplementary active ingredients can also be incorporated into the compositions.

Examples of vehicles include solvents and dispersion media containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), mixtures thereof, and vegetable oils. In many cases, it will be preferred to include isotonic agents, for example, sugars or sodium chloride. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants.

The present invention contemplates the administration of the pharmaceutical compositions described by various routes. Pharmaceutical compositions comprising ApoA1 mimetic polypeptides or fragments thereof of the invention can be administered by any route that ensures bioavailability in the circulation. These routes may include, but are not limited to oral administration, nasal administration, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, transcutaneous administration, administration by inhalation, and intramuscular injection.

Injectable preparations include sterile suspensions, solutions or emulsions of the active substance in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Formulations for injection may be presented in unit dosage form, for example, in ampoules there were in multi-dose containers, and may contain added preservatives.

Alternatively, the injectable formation can be provided in powder form for reconstitution with a vehicle, which includes, but is not limited to, sterile pyrogen-free water, buffer, dextrose solution, etc., before use. To this end, the ApoA1 mimetic polypeptides of the present invention can be lyophilized, or the lyophilized peptide-lipid complex can be prepared. Stored preparations can be supplied in unit dosage forms and can be reconstituted before use in vivo.

For prolonged administration, the active ingredient can be formulated as a prolonged release preparation, for administration by implantation; for example, subcutaneous, intradermal, or intramuscular injection. Therefore, for example, the active ingredient can be formulated with polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives; for example, as a slightly soluble formal of the modified ApoA1 polypeptides or fragments thereof.

Alternatively, transdermal delivery systems manufactured as a disc or adhesive patch that slowly releases the active ingredient for percutaneous absorption can be used. To this end, permeation enhancers can be used to facilitate transdermal penetration of the active ingredient. A particular benefit can be achieved by incorporating the modified ApoA1 polypeptides or fragments thereof of the invention or the peptide-lipid complex into a nitroglycerin patch for use in patients with ischemic heart failure and hypercholesterolemia.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binding agents (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrating agents (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for

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constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or gum arabic); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl p-hydroxybenzoates or sorbic acid). If appropriate, the preparations may also contain buffer salts, flavoring agents, colorants and sweeteners. Preparations for oral administration may be adequately formulated to give controlled release of the active compound.

For oral administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner. For rectal and vaginal administration routes, the active substance can be formulated as solutions (retention enemas), suppositories or ointments.

For administration by inhalation, the active ingredient can be conveniently administered in the form of an aerosol spray presentation from pressurized bases or a memorized, with the use of a propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, dioxide of carbon or other gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for administering a measured amount. Capsules and cartridges can be formulated for example of gelatin for use in an inhaler or insufflator containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

If desired, the compositions may be presented in a container or dispensing device, which may contain one or more unit dosage forms containing the active substance. The package may comprise, for example, metal or plastic foil, such as blister type problems. The package or dispensing device may be accompanied with instructions for administration.

"Unit dosage" formulations are those that contain a dose or sub-dose of the administered ingredient adapted for a particular program administration. For example, "unit dosage" formulations by way of example are those containing a daily dose or unit or daily sub-dose or a weekly dose or weekly unit or sub-dose and the like.

Under usual conditions of storage and use, all preparations of this type should contain preservatives to prevent the growth of microorganisms. Prevention of the action of microorganisms can be prevented with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of stable injectable compositions can be produced by the use of absorption delay agents in the compositions, for example, aluminum monostearate and gelatin.

Before or after formulation, the modified ApoA1 polypeptides should be extensively dialyzed to remove unwanted small molecular weight molecules, and / or lyophilized for easier formulation in a desired vehicle, when appropriate. Sterile injectable solutions are prepared by incorporating the active agents in the necessary amount in the appropriate solvent with several of the other ingredients listed above, if desired, followed by sterilization by filtration. Generally, the dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle containing the basic dispersion medium and the other ingredients required from those listed above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation procedures are vacuum drying and lyophilization techniques that provide a powder of the active ingredient, plus any additional desired ingredient from a previously filtered solution. sterile of it.

"Slow release" pharmaceutical capsules or "sustained release" compositions or preparations can be used and can usually be applied. Slow release populations are generally designed to give a constant drug level for a prolonged period of time can be used to deliver the ApoA1 mimetic polypeptides according to the present invention.

In certain embodiments, liposomes and / or nanoparticles can be used with ApoA1 mimetic polypeptides. Typically, those skilled in the art are aware of the formation and use of liposomes, as summarized below. Liposomes are formed from phospholipids that disperse in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLV)). Generally, MLVs have diameters from 25 nm to 4 µm. The sonication of the MLV results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, which contain an aqueous solution in the nucleus. ApoA1 mimetic polypeptides can also be formulated on phospholipid disks of a size between 8 and 20 nm, through spontaneous reaction with phospholipid liposomes, or through the coolant dialysis procedure.

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The nanocapsules can usually trap compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymeric overload, such ultra fine particles (with a size of approximately 0: 1 µm) should be designed using polymers capable of being degraded in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles satisfying these requirements are contemplated for use in the present invention, and such particles can be easily prepared.

Additional pharmacologically active agents can be supplied together with the primary active agents, for example, the peptides of the present invention. Therapies may include, but are not limited to, simultaneous or sequential administration of the drugs involved. In one embodiment, such agents include, but are not limited to agents that reduce the risk of atherosclerotic events and / or complications thereof. Such agents include, but are not limited to, beta blockers, combinations of beta blockers and thiazide diuretics, statins, aspirin, ace inhibitors, ace receptor inhibitors (ARBs), and the like.

Examples of beta blockers include, but are not limited to cardioselective (selective beta blockers), for example, acebutolol (Sectral), atenolol (Tenormin), betaxolol (Kerlone), bisoprolol (Zebeta), metoprolol (Lopressor), and the like. . Examples of non-selective blockers (beta 1 and beta 2 blockers alike) include, but are not limited to carteolol (Cartrol), nadolol (Corgard), penbutolol (Levatol), pindolol (Visken), propranolol (Inderal), timolol (Blockadren ), labetalol (Normodyne, Trandate), and the like.

Examples of combinations of beta blocker and thiazide diuretic include, but are not limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide, Inderal LA 40/25, Inderide, Normozide, and the like.

Examples of statins include, but are not limited to pravastatin (Pravachol / Bristol-Myers Squibb), simvastatin (Zocor / Merck), lovastatin (Mevacor / Merck), Lipitor (Pfizer), and the like.

Examples of ace inhibitors include, but are not limited to captopril (e.g., Capoten de Squibb), benazepril (e.g., Lotensin de Novartis), enalapril (e.g., Vasotec de Merck), fosinopril (e.g., Monopril de Bristol-Myers), lisinopril (for example, Prinivil de Merck or Zestril of Astra-Zeneca), quinapril (for example, Accupril de Parke-Davis), ramipril (for example, Altace de Hoechst Marion Roussel, King Pharmaceuticals), imidapril, perindopril erbumina (for example, Rhone-Polenc Rorer Aceon), trandolapril (for example, Mavik from Knoll Pharmaceutical), and the like. Suitable ARBS (Ace Receptor Blockers) include, but are not limited to, losartan (e.g., Cozaar de Merck), irbesartan (e.g., Avapro de Sanofi), candesartan (e.g., Astra Merck's Atacand), valsartan (for example, Diovan de Novartis), and the like.

The mimetics of the invention can be used in a method to treat cardiovascular disorders. "Cardiovascular disorder," as used herein, refers to the class of disorders that involve the heart or blood vessels (arteries and veins). In addition "cardiovascular disorder" additionally refers to any disease that affects the cardiovascular system, can be used to refer to those related to atherosclerosis (arterial disease). Cardiovascular disorders may include, but are not limited to, aneurysms, angina, arrhythmia, atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery disease, dilated cardiomyopathy, dysfunction Endocarditis, High Blood Pressure (Hypertension), Hypertrophic Cardiomyopathy, Mitral Valve Prolapse, Heart Attack, Vascular Stenosis and Venous Thromboembolism.

"Arteriosclerosis", as used herein, refers to any hardening (and loss of elasticity) of medium or large arteries (in Greek, "Arterio" means artery and "sclerosis" means hardening), arteriosclerosis is atherosclerosis that It mainly affects the arterioles (small arteries). "Atherosclerosis," as used herein, refers to a hardening of an artery due specifically to an atheroma plaque. Therefore, atherosclerosis is a form of arteriosclerosis. Atherosclerosis is a chronic inflammatory response in the walls of the arteries, due in large part to the deposition of lipoproteins (plasma proteins that carry cholesterol and triglycerides). It is usually called "hardening" or "encrustation" of the arteries. It is caused by the formation of multiple plaques within the arteries.

The method may include the step of administering to a subject a therapeutically effective or biologically effective amount of a pharmaceutical composition that includes an ApoA1 mimetic or fragment thereof that is capable of stimulating cholesterol efflux from cells loaded with lipid. The mimetic of ApoA1 or an amino acid sequence fragment can be modified as described above by replacing all tryptophan with phenylalanine.

Therefore, "biologically effective amounts" or "therapeutically effective amounts", in terms of each of the therapeutic procedures mentioned above are amounts of the at least one modified ApoA1 polypeptide or a fragment thereof effective to exert an anti-inflammatory effect, increased efflux of cellular cholesterol, or improvement of symptoms of cardiovascular disease.

"Administration," as used herein, means provision or administration of a composition that includes at least one ApoA1 mimetic polypeptide or a fragment thereof in an amount (s) and during a

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period of time (s) effective for exerting an anti-inflammatory effect, increased efflux of cellular cholesterol, or improvement of cardiovascular disease symptoms. In general, passive administration of protein therapeutic agents is preferred, in part, for their simplicity and reproducibility.

Due to the properties of the oxidation resistant polypeptides of the present invention, the ApoA1 mimetic polypeptides and fragments thereof described herein can also be used in a method to stimulate cholesterol efflux from a cell to the liver. The method includes the step of administering to the cell a biologically effective amount of purified polypeptide having an amino acid sequence corresponding to a part of SEQ ID NO: 1, wherein X is phenylalanine.

The therapeutic methods and uses of the invention also extend to the provision of nucleic acids encoding at least one therapeutic agent that includes ApoA1 mimetic polypeptide (s) in an effective manner to result in its expression in the vicinity of the symptom, condition or disease directed. Any genetic therapy technique, such as naked DNA administration, recombinant genes and readers, cell based administration, including ex vivo manipulation of patient cells, and the like can be used.

It will also be understood that even in such circumstances where the dose, or combined therapy of ApoA1 polypeptides are at the lower end of the intended therapeutic range, it may happen that this therapy is still equally or even more effective than all other therapies known in the context of the particular disorder or patient. Unfortunately, it is clear to a clinical professional that certain disorders and conditions cannot be treated effectively in an intermediate or long term, but this does not invalidate the usefulness of the present therapy, particularly when it is at least approximately as effective as the others. Proposed strategies usually.

Generally, the intention of the therapeutic regimens of the present invention is to produce a significant increase in cholesterol efflux, while the dose is still maintained below the levels associated with an unacceptable toxicity. In addition to varying the dose itself, the administration regimen can also be adapted to optimize the treatment strategy.

The active agents of the present invention are also useful in a number of contexts. For example, it has been observed in cardiovascular disorders (eg, atherosclerosis, stroke, etc.). Therefore, in certain embodiments, the present invention contemplates the administration of one or more of the active agents described herein to a subject at risk of, or contracting a symptom of atherosclerosis and / or an associated pathology (for example, stroke).

Therefore, for example, one or more pharmaceutical compositions of the present invention can be administered prophylactically to a person who is at risk of coronary disease during the flu season. In another specific example, post-myocardial infarction of a subject could be treated by i.v. to reduce plate size and stabilize plate to prevent breakage or erosion.

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should note that the techniques disclosed in the examples that follow represent techniques discovered by the inventor that work well in the practice of the invention, and therefore may be considered to be preferred modes of practice. However, those skilled in the art should observe, in view of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain a similar or similar result.

Examples

In the following examples, the inventors sought to determine the effects of the modification of lysine, methionine, and tryptophan, which are MPO-sensitive residues in ApoA1. It was found that the substitution of the four tryptophan residues in ApoA1 with leucines leads to a loss of function, while the replacement of tryptophan with phenylalanine not only retains the function of ApoA1, but also makes it resistant to oxidative inactivation with MPO.

Procedures

Mass spectrometry

ApoA1 obtained from human atheroma was isolated by immunoaffinity chromatography as described above. ApoA1 was eluted in glycine buffer (pH 2.5) and was directly digested with trypsin or first separated with SDS-PAGE and subjected to digestion with trypsin gel. Mass spectrometry was performed and collision induced dissociation spectra (CID) were obtained, as described above. Chlorotyrosine and 2-amino adipic acid analyzes were performed after acid hydrolysis with internal standards of heavy isotopes as described above using duplicate ApoA1 assays from human atheroma or from plasma from healthy volunteers isolated by immunoaffinity chromatography. .

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Mutagenicity · Directed to the Site and Production of ApoA1 Recombinants

Point mutations were prepared for tryptophan residues (8, 50, 72, 108) and methionine (86, 112, 148) using Stratagene's Quick-Change Mutagenesis Kit and confirmed by DNA sequencing. Plasmids were transformed into ApoA1 expression of strains BL21 (DE-3) pLysS, Escherichia coli lnJ and purification was performed as described above. Extensive dialysis of rh-ApoA1 was performed with respect to PBS or MPO reaction buffer (60 mmol / l sodium phosphate, 100 mmol / l sodium chloride, 100 µmol / l diethylenetriamine pentaacetic acid, pH 7.0 ) to remove any trace of imidazole, it was analyzed by SDS-PAGE, and found to be pure at> 95%. Since the substitution of Trp and Met alters the OD280 of the protein and the reactivity with respect to the BCA or Lowry protein assays, the protein concentrations were determined based on free amines using the o-phthaldialdehyde (OPA) assay, with a ApoA1 pattern derived from human plasma (Biodesign), as described above. The cleavage of the initial Met and His tag from rh-ApoA1 was performed by treatment with formic acid, followed by purification with rapid liquid protein chromatography (FPLC).

Lysine modifications of ApoA1

ApoA1 derived from human plasma was dialyzed with respect to PBS and diluted to 0.5 mg / ml. Reductive methylation with lysine was performed as described above. The extent of lysine modification was determined by the OPA test. Next, ApoA1 was dialyzed with respect to MPO reaction buffer, and the protein concentration of ApoA1 modified with lysine was determined using BCA reagents.

Modifications of ApoA1 with MPO and Hypochlorous Acid

The MPO at a final concentration of 57 nmol / l, prepared as described above, was added to ApoA1 at 100 µg / ml (3.5 µmol / l) which had been extensively dialyzed with respect to MPO reaction buffer. The reaction began by adding hydrogen peroxide at varying molar ratios with respect to ApoA1 in 4 aliquots at 15 minute intervals at 37 ° C, and incubation continued for 90 minutes, at which time 2 mmol / l of L was added. -methionine inactivate the reaction. For the chemical modification of ApoA1, sodium hypochlorite (NaOCI) was added to 100 µg / ml of ApoA1 in MPO buffer at varying concentrations in 4 aliquots at 15 minute intervals at 37 ° C. After a total incubation time of 60 minutes, 2 mmol / l of L-Methionine was added to stop the reaction.

ABCA I Dependent Cholesterol Efflux Assay

RAW 264.7 murine macrophage cells were labeled with [3H] cholesterol and treated with 0.3 mmol / l of 8Br-cAMP to induce ABCA1 activity, as described above. The cells were washed and followed for 4 hours in serum free medium in the presence of 0.3 mmol / l of 8Br-cAMP a and in the presence or absence of various ApoA1 preparations. Radioactivity in the monitoring media was determined after a brief centrifugation to the sedimented residue. The radioactivity in the term cells by extraction in hexane: isopropanol (3: 2) with the solvent evaporated the scintillation world before counting. The percentage of cholesterol efflux was calculated as 100 X (dpm in the middle) / (dpm in the middle + cell dpm).

Lipid Binding Activity Test

The lipid binding activity of ApoA1 was evaluated through inhibition of phospholipase C-mediated aggregation of human low density lipoprotein, will be performed as described above. The inventors have previously shown that this test gives similar results to those observed with the DMPC dispersion removal test, but is more sensitive and requires less ApoA1. The final concentration of ApoA1 used in this assay was 12.5 µg / ml which was sufficient to decrease the initial rate LDL aggregation by ≈75%.

ApoA1 Reticulation Detection

250 ng of ApoA1 were denatured per lane in a SDS demonstration buffer, developed on a 10% Tris glycine gel in the presence of SDS; and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed sequentially with goat anti-human ApoA1 primary antibody (dilution

1: 1,000 DiaSorin) and rabbit anti-goat HRP conjugate antibody (1: 1,000 dilution), and ApoA1 was visualized with a substrate with increased chemiluminescence.

Results

Modifications of ApoA1 in Human Atheroma

The inventors determined whether ApoA1 isolated from human atheroma cells included modified tryptophan, methionine and lysine residues Using tandem mass spectrometry, the inventors were able to detect monohydroxytryptophan residues in the four tryptophan positions, 8, 50, 72, and 108, and dihydroxytryptophan at position 108 within ApoA1 (Figs. 1A-F). The inventors previously identified monoy di-hydroxytryptophan in the W72 moiety in ApoA1 modified with MPO in vitro (Peng, D.Q., Wu, Z., Brubaker, G.,

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receptor of ApoA1 cholesterol, since the 4WL variant per day in most of this activity in cholesterol efflux studies conducted with respect to a wide range of ApoA1 doses, while the 4WF variant retained this activity (Fig .5A). The inventors examined the predicted alpha helix content of these proteins by CD using the K2d algorithm, and found that the wild-type protein had 57% alpha helix, while the 4WL and 4WF variants both had increased the helix contents. alpha in 79% and 77%, respectively. Therefore, the loss of efflux and the lipid binding activity of the 4WL variant cannot be attributed to the loss of helical content.

Both rh-ApoA1 and the 4WF variant were subjected to the MPO / CL- / H2O2 oxidation system at increasing doses of H2O2. Fig. 5B shows the result of a representative study of 4 different experiments using two independent preparations of each protein. As noted above, the ABCA1-dependent cholesterol acceptor activity of wild-type ApoA1 was inhibited by increasing oxidation induced by MPO; however, the 4WF variant maintained this activity even at a H2O2: ApoA1 ratio of 15.

The MPO / Cl / H2O2 oxidation system generates HOCl, the active reagent of the bleach, and the inventors and others have previously shown that ApoA1 treatment with HOCl results in a loss of cholesterol acceptor and lipid-binding activity. Therefore, the inventors subjected the wild-type ApoA1 and the 4WF variant to increasing doses of HOCl. As with the findings of the MPO modification system, the cholesterol acceptor activity of the 4WF variant was resistant to this treatment while the efflux activity of wild-type rh-ApoA1 was altered by the increase in dose of HOCl (Fig. 6).

The lipid binding activity of rh-ApoA1 and the 4WF variant were evaluated by an DMPC emulsion removal assay, and both proteins showed an equivalent activity (Fig. 7A). Lipid-binding activity without rh-ApoA1 4WF cells was also resistant to MPO-mediated inhibition, compared to rh-ApoAl (Fig. 4B) using a PLC-mediated LDL aggregation assay (Fig. 7B).

Modification of ApoA1 with MPO leads to extensive crosslinking that results in dimers, multimers, and also supposedly intramolecular crosslinks. The inventors have previously shown that the MPO-mediated ApoA1 crosslinking pattern was not altered in the variant with the 7 tyrosine residues converted to phenylalanine. After subjecting the rh-ApoA1 and the 4WF variant to oxidation with MPO / Cl- / H2O2 at increasing doses of H2O2 (using the identical protein products that were used for the flow in Fig. 5B), the inventors observed an alteration of the migration of both proteins in denaturing gels consistent with intermolecular crosslinking. However, the migration patterns were different, with the 4WF variant giving a predominantly acute band of approximately 70 kD, while the wild-type protein provided a predominantly less marked zone between 55 and 65 kD (Fig. 8). Monomer migration was altered for both proteins, which could be indicative of intramolecular crosslinks or amino modifications. Although the 4WF variant is resistant to loss of MPO-mediated cholesterol acceptor activity, this variant was more susceptible to MPO-induced crosslinking, in particular at low doses of H2O2. The inventors also subjected these modified proteins to structural analysis with CD, and found that both were susceptible to loss of alpha helix content, although the 4WF variant began with a higher value.

The inventors also prepared rHDL by choke dialysis using POCP and wild-type or 4WF ApoA1. Both provided a similar pattern of rHDL discs calculated by non-denaturing gels at ~ 9.8, 12, and 17 nm, without any ApoA1 without remaining liquid (Fig. 9). The inventors tested wild-type and 4WF rHDL, and both were equally competent to mediate cholesterol efflux independent of ABCA1 from RAW264.7 cells (Fig. 10), without ABCA1-dependent acceptor activity, such as It was expected for ApoA1 fully lipidated.

The inventors synthesized the amphipathic helical p18 peptide described above, which contains a tryptophan moiety in position 2. The inventors also synthesized analogs that replace tryptophan with phenylalanine (P 18 WF) or leucine (P 18 LF). The inventors demonstrated that these tryptophan-free peptides have no dose-dependent ABCA1-mediated cholesterol reading activity (Fig. 11).

This disclosure also contemplates the following:

one.

A purified polypeptide comprising an ApoA1 mimetic that is capable of stimulating cholesterol efflux from cells loaded with lipid, ApoA1 mimetic having an amino acid sequence that includes at least a portion of the ApoA1 amino acid sequence or a mimetic of ApoA1 containing at least one tryptophan, at least one tryptophan from ApoA1 or mimetic of ApoA1 that is being substituted with an oxidation resistant amino acid in the amino acid sequence of the ApoA1 mimetic.

2.

 The polypeptide of clause 1, wherein the ApoA1 mimetic is resistant to at least one oxidation with MPO generated oxidant MPO, oxidation with a kind of reactive chlorination generated by MPO, oxidation associated with the MPO / H2O2 / Cl system -, oxidation with HOCl / OCl-, oxidation with a reactive nitrogen species generated by MPO, oxidation associated with the MPO / H2O2NO2- system, oxidation with nitrogen dioxide, oxidation with peroxynitrite (ONOO-), oxidation with peroxycarboxynitrite (ONOOCO2- ), or oxidation with

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a product formed when ONOO-acts in the presence of CO2 or HCO3-in buffer.

3.

 The polypeptide of clause 1, phenylalanine being the amino acid resistant to oxidants.

Four.

The polypeptide of clause 1 comprising at least a part of SEQ ID NO: 1, wherein X is selected from the group consisting of tryptophan or phenylalanine and at least one X is phenylalanine.

5.

The polypeptide of clause 1, mimetic of ApoA1 consisting essentially of about 5 to about 50 amino acids of native ApoA1, which includes at least one tryptophan, and the at least one tryptophan is substituted with an oxidant resistant amino acid.

6.

The polypeptide of clause 1, mimetic of ApoA1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69, wherein X is a tryptophan or an oxidizer resistant amino and at least one X It is replaced by an oxidant resistant moiety.

7.

 The polypeptide of clause 1, a polypeptide comprising at least one amino acid residue "D".

8.

The polypeptide of clause 1, in which all or most of the enantiomeric amino acids are "D" amino acids.

9.

 The polypeptide of clause 1, a polypeptide further comprising a protective group.

10.

 The polypeptide of clause 1 further comprising a polyhistidine tag.

eleven.

 The polypeptide of clause 1, a polypeptide that is resistant to oxidative inactivation by inactivation with myeloperoxidase (MPO) when administered to a subject.

12.

 The polypeptide of clause 1, a polypeptide that is a fusion protein.

13.

A purified apolipoprotein polypeptide comprising a modified native apolipoprotein amino acid sequence by replacing one or more tryptophan moieties of the native amino acid sequence with an oxidant resistant amino acid.

14.

A pharmaceutical composition comprising the polypeptide of clause 1, and a pharmaceutically acceptable excipient.

fifteen.

The pharmaceutical composition of clause 14, wherein the composition is formulated for administration to a subject by a route selected from the group consisting of oral administration, nasal administration, rectal administration, intraperitoneal injection, intravascular injection, subcutaneous injection, administration transcutaneous, administration by inhalation, and intramuscular injection.

16.

 A procedure to treat cardiovascular disorders comprising the stage of:

administering to a subject an ApoA1 mimetic that is capable of stimulating cholesterol efflux from cells loaded with lipid, ApoA1 mimetic that has an amino acid sequence that includes at least a part of the ApoA1 amino acid sequence or a mimetic of the ApoA1 containing at least one tryptophan, at least one tryptophan from ApoA1 or mimetic of ApoA1 that is being substituted with an oxidant resistant amino acid in the amino acid sequence of the ApoA1 mimetic.

17.

The procedure of clause 16, in which the mimetic of ApoA1 is resistant to at least one oxidation with oxidant generated by MPO MPO, oxidation with a kind of reactive chlorination generated by MPO, oxidation associated with the MPO / H2O2 / Cl system -, oxidation with HOCl / OCl-, oxidation with a reactive nitrogen species generated by MPO, oxidation associated with the MPO / H2O2 / NO2- system, oxidation with nitrogen dioxide, oxidation with peroxynitrite (ONOO-), oxidation with peroxycarboxynitrite ( ONOOCO2-), or oxidation with a product formed when ONOO-acts in the presence of CO2 or HCO3-in buffer.

18.

 The procedure of clause 16, phenylalanine being the amino acid resistant to oxidants.

19.

The method of clause 16 comprising at least a part of SEQ ID NO: 1, wherein X is selected from the group consisting of tryptophan or phenylalanine and at least one X is phenylalanine.

twenty.

The procedure of clause 16, mimetic of ApoA1 consisting essentially of about 5 to about 50 amino acids of native ApoA1, which includes at least one tryptophan, and the at least one tryptophan is substituted with an oxidant resistant amino acid.

twenty-one.

The method of clause 16, mimetic of ApoA1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69, wherein X is a tryptophan or an oxidizer resistant amino and at least one X It is replaced by an oxidant resistant moiety.

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22

The procedure of clause 16, a polypeptide that is resistant to oxidative inactivation by inactivation with myeloperoxidase (MPO) when administered to the subject.

2. 3.

 A procedure to stimulate the efflux of cholesterol from a cell comprising the stage of:

administer to the cell a biologically effective amount of an ApoA1 mimetic that is capable of stimulating cholesterol efflux from cells loaded with lipid, ApoA1 mimetic having an amino acid sequence that includes at least a part of the amino acid sequence of ApoA1 or an ApoA1 mimetic that contains at least one tryptophan, at least one ApoA1 tryptophan or ApoA1 mimetic that is being substituted with an oxidant resistant amino acid in the amino acid sequence of the ApoA1 mimetic.

24.

The procedure of clause 23, a polypeptide that does not substantially inhibit apolipoprotein lipid binding and cholesterol efflux.

25.

The procedure of clause 23, in which the mimetic of ApoA1 is resistant to at least one oxidation with oxidant generated by MPO MPO, oxidation with a kind of reactive chlorination generated by MPO, oxidation associated with the MPO / H2O2 / Cl system -, oxidation with HOCl / OCl-, oxidation with a reactive nitrogen species generated by MPO, oxidation associated with the MPO / H2O2 / NO2- system, oxidation with nitrogen dioxide, oxidation with peroxynitrite (ONOO-), oxidation with peroxycarboxynitrite ( ONOOCO2-), or oxidation with a product formed when ONOO-acts in the presence of CO2 or HCO3-in buffer.

26.

 The procedure of clause 23, phenylalanine being the amino acid resistant to oxidants.

27.

The method of clause 23 comprising at least a part of SEQ ID NO: 1, wherein X is selected from the group consisting of tryptophan or phenylalanine and at least one X is phenylalanine.

28.

The procedure of clause 23, mimetic of ApoA1 consisting essentially of about 5 to about 50 amino acids of native ApoA1, which includes at least one tryptophan, and the at least one tryptophan is substituted with an oxidant resistant amino acid.

29.

The method of clause 23, mimetic of ApoA1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69, wherein X is a tryptophan or an oxidant resistant amino and at least one X It is replaced by an oxidant resistant moiety.

30

A procedure to improve one or more symptoms of an inflammatory condition in a subject comprising:

administer to the subject an ApoA1 mimetic that is capable of stimulating cholesterol efflux from cells loaded with lipid, ApoA1 mimetic that has an amino acid sequence that includes at least a part of the amino acid sequence of ApoA1 or an ApoA1 mimetic which contains at least one tryptophan, at least one tryptophan from ApoA1 or mimetic of ApoA1 that is being substituted with an oxidant resistant amino acid in the amino acid sequence of the ApoA1 mimetic.

31.

 The procedure of clause 30, the inflammatory condition comprising atherosclerosis.

32

 The procedure of clause 30, the inflammatory condition comprising stenosis.

33.

 The procedure of clause 30, further comprising the step of administering a statin to the subject.

3. 4.

The procedure of clause 30, wherein the ApoA 1 mimetic is resistant to at least one oxidation with oxidant generated by MPO MPO, oxidation with a kind of reactive chlorination generated by MPO, oxidation associated with the MPO / H2O2 / system Cl-, oxidation with HOCl / OCl-, oxidation with a reactive nitrogen species generated by MPO, oxidation associated with the MPO / H2O2 / NO2- system, oxidation with nitrogen dioxide, oxidation with peroxynitrite (ONOO-), oxidation with peroxycarboxynitrite (ONOOCO2-), or oxidation with a product formed when ONOO-acts in the presence of CO2 or HCO3-in buffer.

35

 The procedure of clause 30, phenylalanine being the amino acid resistant to oxidants.

36.

The method of clause 30 comprising at least a part of SEQ ID NO: 1, wherein X is selected from the group consisting of tryptophan or phenylalanine and at least one X is phenylalanine.

37.

The procedure of clause 30, mimetic of ApoA1 consisting essentially of about 5 to about 50 amino acids of native ApoA1, which includes at least one tryptophan, and the at least one tryptophan is substituted with an oxidant resistant amino acid.

38.

The method of clause 30, mimetic of ApoA1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69, wherein X is a tryptophan or an amino

Claims (1)

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